The importance of sensor based bioanalytical methods and instruments has been increasing the last couple of years in the sectors of biotechnology and medical research as well as in pharmacological research. The main reason lying in the increasing demand of fast analytical methods that yield quantitative data on biomolecular interactions.
Optical affinity sensors deal with these demands in an ideal way, as they are able to detect without delay, in real time, biomolecular binding events without the utilization of interfering labels.
The arising of highly parallel batches for the analysis of complex nucleic acid- or protein-mixtures, as well as the rising use of combinatorial synthesis procedures within the pharmaceutical active substance search, make the high throughput compatibility of following methods of analysis a central criterion. This need could be favorably covered by optical sensors, which can measure many bonding reactions in parallel. In contrast to the devices available today, such sensors would have to be able to analyze the entire image of a sensor array instead of only a few individual measuring points.
Several different state of the art optical detection principles are well-known which can be used for the real time analysis by biomolecular interactions.
Most procedures use the changes of refractive indices due to binding reactions with the sensor surface.
Most common probably the already explained surface plasmon resonance—SPR sensors, which can be implemented apparatively relatively easily.
The shift of the SPR minimum is normally measured spectrally or, more commonly, angularly resolved.
The spectrally resolved detection, which normally is not as sensitive as the angularly resolved one, is advantageously used in cases in which an angularly resolved detection is not apparatively applicable.
One example is the fiber optic SPR (WO 94/16312 A1), in which light from a broadband source is coupled into a gold-coated optical fiber and the shift of the resonance wavelength is measured.
The angularly resolved detection is described, for example, in WO 90/05305. In this apparatus, a metal film is illuminated with convergent light beams, and the angle shift is observed by means of a diode array/lens system-combination.
Such a device demands a relatively large, mechanically very massive measuring head, which makes such an apparatus lavish. A apparativly simpler variant, as described in DE 19817472, only uses two photodiodes to determine the SPR minimum shift, making this apparatus a little bit simpler.
A principally different principle is described by Kooyman et al. (R. P. H. Kooyman, A. T. M. Lenferink, R. G. Eenink and J. Greve (1990) Anal. Chem. one, 63, pp. 83-85). Here the angle of the incident laser beam is varied over time with a scanner mirror and the corresponding change of intensity of the reflected light is detected by means of a photoelectric cell. The system described there supplies good results when measuring few points and is relatively unelaborate.
Other detection principles comprise, for example, the Resonant Mirror (Cush, R., Cronin, J. M., Goddard, N. J., Maule, C. H., Molloy, J. und Stewart, W. J. (1993) Biosensors & Bioelectronics, 8, pp. 347-353), the integrated optical interferometer (DE 4033357), the difference-interferometer (Fattinger, Ch., Koller, H., Schlatter, D. und Wehrli, P. (1993) Biosensors & Bioelectronics, 8, pp. 99-107), the grating coupler (Tiefenthaler, K. (1992) Advances in Biosensors, Vol. 2, pp. 261-289) or the Reflectometric Interference Spectrometer (DE 19615366 A1).
The production of the exchangeable sensor is clearly more complex in all these enumerated techniques than with the SPR, this being one of the reasons, besides others, for them being inferior to the SUPERCHARGER.
All procedures specified above have in common that they do not work spatially resolved and thus cannot cope with multiple analytes.
In past years several methods were therefore developed, which make parallel measuring possible on different parts of the sensor chip.
In this regard, a further development of the above already mentioned grating coupler is described, e.g., in WO 95/03538 or EP 1031828 A1; a spatially resolved reflectrometric interference spectrometer is known from DE 19828547 A1.
Apart from the disadvantage of the complex manufacturing of the exchangeable sensors, these systems also have the disadvantage that they divide the sensor surface into discrete and relatively large parts and the devices therefore become either quite large or exhibit a limited capacity.
As the SPR sensors are technically easier to implement and theoretically allow a nearly arbitrarily small partitioning of the sensor surface, clearly more implementation solutions exist. The first picture-giving SPM (Surface Plasmon Microscope) was developed in 1988 (Knoll, W. and Rothenhaeusler, B. (1988) Nature, 332, pp. 615-617).
In this and other well-known procedure (DE 19829086, as well as Frutos, A. G. and Corn, R. M. (1998) Anal. Chem., July 1, pp 449A-455 A) a widened laser beam is radiated on a metal surface at a fixed angle and the changes of intensity of the picture reflected on a CCD camera is evaluated. The main disadvantage of this method is that only intensity changes of the pixels are recorded and not the angles of the SPR minima.
This results in a clearly worse sensitivity and a strongly reduced dynamic range. In addition, some changes of individual brightness values might be ambiguous under some conditions—it then cannot be determined in which direction the SPR minimum is shifted.
An improvement of the described SPM technology is revealed in DE 3909144. A picture of the sensor surface is recorded using different incidence angles and the SPR minimum angles for up to 5×5 μm small surface sections are determined with downstream image processing. Although quite a high accuracy can be obtained with this procedure in principle, incidence and reflection angles must both be changed for imaging, which is mechanically complex and which can only be realized using a low data acquisition frequency. A two-dimensional fast real time analysis of bonding reactions on the chip surface is therefore not possible with this arrangement.
A spatially resolved SPR sensor with spectral detection is well-known from WO 00/22419. However, it uses mobile hole or slit apertures, in order to successively light up different ranges of the sensor surface, increasing the mechanical complexity, slowing down the data acquisition frequency and setting the size of the individual measuring points to a fixed value from the beginning on.
An angularly resolved SPR equipment with spectral detection is described in WO 99/30135. For the utilization as imaging sensor the use of a mask or a lens array is suggested. The disadvantages of this arrangement closely correspond to those of the sensor mentioned in the preceding section.
A system with mechanical change of the incidence angle and likewise mechanical change of the XY position of the measuring point on the sensor chip is known from WO 00/46589. Unfavorable are, above all, the complex structure and large mobile mechanical components, which entail a low data acquisition frequency.
Moreover, EP 0973023 describes a compact SPR transducer with angle resolved detection. The measuring range and the detector array are here divided into several areas, for which separate SPR signals are recorded. The areas of the individual sensitive regions are determined by the size of the transducers and are thus relatively large. A real high throughput ability might therefore only limits the application as a biosensor.
WO 98/34098 describes a spatially resolved SPR sensor with a complex lens and mirror system for the synchronously detection of the SPR minimum angles for a multiplicity of pixels. A relatively high measuring frequency can be realized using this scheme, but it also is a very complex contraption.